This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. This is a 2-year proposal for APS beam time for structural studies of biological macromolecules. It focuses on the mammalian serine protease thrombin, on two flavoprotein redox systems and on electron transfer (ET) mutants of a cupredoxin that accepts electrons from a quinoenzyme. Human thrombin catalyzes the proteolytic conversion of fibrinogen to fibrin, the major component of a blood clot. The structures of several mutant forms of these proteins, as well as of various enzyme-substrate and enzyme-inhibitor complexes will be determined in order to elucidate their modes of action. These studies will be extended to the murine enzyme which differs in several respects from the human enzyme. One of the redox flavoproteins, heterotetrameric sarcosine oxidase (TSOX), isolated from Pseudomonas maltophilia, contains FAD, FMN and binds tetrahydrofolate (THF). TSOX catalyzes the oxidation of sarcosine (N-methyl glycine) to an imine intermediate and reduced FAD;in a second step the imine intermediate combines with THF to form 5,10-methylene-THF. The two catalytic sites are separated by about 35 A. The FAD is reoxidized by ET to FMN and the latter then reduces molecular oxygen to yield peroxide. A second redox flavoenzyme is nikD, which catalyzes an early step in the biosynthesis of nikkomycin antibiotics in Streptomyces tendae. It is a monomer of 45 kDa containing FAD;its structure is known in two distinct conformation. Structures of its mutants will be studied. Amicyanin is a blue copper protein of 9 kDa that accepts electrons from methylamine dehydrogenase (MADH) in Paracoccus denitrificans. The redox properties of several amicyanin mutants show significant differences from the wild type protein;atomic resolution structures of these mutants are known. Crystals of ET complexes of these mutants with MADH will be studied.